The rapid emergence of antibiotic resistance, and decline in discovery of new antibiotics, has created a significant gap in the effective management of microbial infections. Additionally, persistent use of antibiotics in agriculture has further increased antibiotic resistant bacteria strains, thus making antibiotic resistant microbial infections an acute public health concern. According to the WHO global surveillance report for antimicrobial resistance, very high rates of resistance have been observed in common bacterial strains such as Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus (apps.who.int/iris/bitstream/10665/112642/1/9789241564748_eng.pdf?ua=).
E. coli related urinary tract infections (UTIs) are among the most frequently encountered bacterial infections in the United States (1,2) with an annual incidence of over 8 million. Infection may involve both lower (bladder, cystitis) and upper urinary tract (kidney, pyelonephritis) and lead to significant morbidity, including permanent kidney damage (chronic pyelonephritis) or life threatening bacterial sepsis. The cumulative morbidity is profound with over 100,000 hospitalizations per annum. (3)
Appropriate treatment of UTI is critical for optimal outcome and requires use of agents that are active against the infecting pathogen. In the outpatient setting, patients are often prescribed empiric antibiotic therapy as susceptibility patterns of uropathogens have, until recently, been generally predictable. In hospitalized patients, empiric therapy is given for 48-72 hours until traditional culture results and susceptibility data are available. However, the rapid emergence of antibiotic resistance in recent years presents a significant challenge for UTI management.(4-8) In particular, carbapenem-resistant and extended-spectrum β-lactamase producing Enterobacteriaceae are an immediate public health threats, as they express resistance to commonly used empiric antimicrobial regimens. (9-11) Pathogens amongst the ESKAPE group are predicted to become resistant to currently available antibiotics within the next decade. Alarmingly, these are amongst the most common agents of UTI seen in hospitalized patients.
Initiation of active, appropriate antimicrobial patient therapy is ultimately dependent upon turnaround time of infection diagnostic methods. (12) Historically, this testing has relied on routine culture to isolate organisms; biochemical tests for bacterial identification; and culture based antibiotic susceptibility testing (AST). (13) However, using traditional technology, susceptibility results are generally not available for 48-72 hours. This means that for the first three days of suspected infection, therapy is largely unguided. Therefore, the ability to shrink this window by rapidly performing AST for bacterial pathogens is urgently needed. Rapid AST will also decrease emergence of resistance by allowing targeted (antibiotic de-escalation) or discontinuation of therapy if bacterial infection is ruled out (14,15).
New technologies have been applied to address this problem (16). For example, MALDI-TOF mass spectrometry (MS) and nucleic acid amplification tests (NAATs) are beginning to transform the clinical microbiology laboratory. These newer platforms offer several benefits over traditional culture methods; however, they also have significant limitations. MS is generally rapid and can identify a large database of bacteria. However, lengthy culture pre-amplification is still required, since analysis can only be performed on isolated bacterial colonies. (17) Also, MS-based AST only marginally decreases the time needed to identify organisms and does not address the need for rapid AST. Lastly, MS equipment is expensive ($200,000-300,000), bulky, complex to operate, and requires regular maintenance. (16) Therefore, MS systems must be placed in a centralized laboratory, away from the site of patient care, further delaying access to diagnostics.
Alternatively, NAATs have the capability to identify specific organisms or resistance genes directly from a clinical sample within two hours, including sample preparation. (18-21) However, they fall short in their ability to yield a comprehensive resistance profile. Specifically, resistance mechanisms are often multifactorial. Therefore, even highly multiplexed NAATs cannot accurately predict phenotypic susceptibility. (22) For example, resistance to a carbapenem in Gram negative bacilli (GNB) may reflect contributions from multiple β-lactamases, porins, and efflux pumps. Indeed, a GNB producing the New Delhi metallo-β-lactamase 1 (NDM-1) enzyme may carry up to eight plasmids (5) together encoding scores of resistance elements that may interact in unexpected ways. (23) Therefore, the Clinical and Laboratory Standards Institute (CLSI) now recommends using carbapenem MIC as the sole criterion for assessing susceptibility, as the mere presence or absence of a CRE genetic element alone does not predict phenotypic response.
Heterogeneity in single cell responses arises from intrinsic stochasticity in both transcription and translation, thereby leading to significant variability in quantitative levels of mRNA and protein within cell populations. This results in biological noise, which can be further enhanced by minor differences in environmental stimuli, variations in cell state and polyfunctional responses. This heterogeneity is an essential characteristic of cellular systems and must be assessed by analyzing individual cell behavior. Furthermore, the dynamic nature of biological processes occurs at varying time scales, requiring continuous real-time evaluation of single cell outcomes. This is particularly evident in analysis of immune responses, which involve a variety of cell types.
Currently, flow cytometry is the most useful technique for single cell analysis, due to its high-throughput and multiplexing capability. However, it cannot provide time-varying spatiotemporal resolution of signaling dynamics in the same cell. Other single cell analysis techniques include laser scanning cytometry, capillary electrophoresis and laser capture microdissection. Many of these techniques suffer from limitations of throughput and complicated operation.
Microfluidic single cell analysis tools have emerged as a powerful alternative to conventional cell culture techniques with respect to throughput, multiplexing, sensitivity, and robust control of cellular microenvironment. Single cells have been captured by valve-based methods (67), dielectrophoretic mechanisms (68,69), and optical tweezers (70). However, active mechanisms such as dielectric forces can negatively impact cell viability; additionally, the throughput achieved with these methods is generally low. Microwells utilize passive gravity-based methods to allow single cell sedimentation followed by stimulation of cells (71, 72). However, non-adherent cells could potentially be lost from their holding sites over time with this technology. Another commonly implemented method relies on manipulating fluid flow or employing hydrodynamic guiding features to direct cells towards traps, thus allowing cells to be sequestered in variously shaped docking structures (73-76). Hydrodynamic arrays have been extensively investigated to achieve optimal capture efficiency and single cell compartmentalization by assessing various trap structure, position and distance (77-80). However, a common limiting feature of most of these microfluidic approaches is the lack of isolation of a cell from its neighbors, leaving room for paracrine effects. Thus, there remains a need for new methods that allow cell function and cell-cell interactions to be analyzed in isolation.